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1. Technical Field
The field of this invention is structural biology.
2. Background of the Invention
The identification of the three-dimensional structure of proteins and other biological molecules is of intense interest in biology, pharmacology and related fields (both academic and industrial), as the three-dimensional structure can have a profound effect on biological activity, e.g. binding characteristics, catalytic activity, etc. Accordingly, much effort has been expended in the development of methods for determining the three-dimensional structure of biological particles of interest, such as proteins.
Electron microscopy has been widely employed in structure determinations of biological specimens. While electron microscopy has been employed with some success to determine the structure of proteins, electron microscopy of biological specimens is limited in resolution by beam-induced specimen damage because single organic molecules are destroyed by electron irradiation sufficient to reveal structural details. In addition to damaging the specimen, even low electron doses impair the quality of electron imaging by causing localized heating, specimen movement, and specimen charging. These difficulties can be overcome by image averaging and optimized data collection techniques.
Image averaging to improve the signal-to-noise ratio of low-dose electron micrographs has been accomplished by diffraction from ordered arrays of molecules or by computational methods of aligning the images of single particles. While the diffraction approach has yielded structural information for several specimens, the necessity of forming a crystalline specimen is a severe impediment, as it prevents the study of a great many biological objects, including partially irregular or inhomogeneous molecules and molecular complexes. The very large multi-protein complexes of most biological interest are especially prone to these limitations.
Escape from the requirement for crystals by computational alignment of single particles relies on the detection of image details to determine the relative orientations of the particles and permit image averaging. The very paucity of detail in a low dose electron micrograph that necessitates averaging unavoidably limits the precision of alignment for the purpose of averaging. Alignment in currently employed protocols is enhanced by recording images at high defocus values, which results in a loss of contrast at high resolution.
As such, there is continued interest in the development of new methodologies for determining the three dimensional structure of proteins and other biological particles. Of particular interest would be the development of a cryoelectron microscopy protocol that did not suffer from the disadvantages from which current cryoelectron microscopy protocols suffer, such as low resolution, high computational demands, and excessive human demands.
Articles of interest include: Jensen and Komberg, Proc. Nat""l Acad. Sci. USA (August 1998) 95: 9262-9267; Boisset et al., Structural Biology (1992) 109:39-45; Starink et al., Biophysical Journal (May 1995) 68:2171-2180; Wagenknecht et al., Biophysical Journal (December 1994) 67:2286-2295; and Wilkens et al., Arch. Biochem. Biophys. (Nov. 15, 1992) 299:105-109. Also of interest are: Ribroux et al., J. Histocytochemistry and Cytochemistry (1996) 44: 207-213; Thygesen et al., Structure (May 15, 1996) 4:513-518; and Lyons et al., Protein Engineering (1990) 3:703-708. Additional articles disclosing cryoelectron microscopy methods include: Radermacher et al., Biophys J. (1992) 61:936-940; Gogol et al., Biochemistry (1989)28:4709-4716; and Gogol et al., Biochemistry (1989) 28:4717-4724.
Methods and compositions for determining the three dimensional structure of a particle are provided. In the subject methods, at least four heavy atom clusters are rigidly attached to the particle of interest. An image of the heavy atom cluster labeled particle is then obtained by electron microscopy. The obtained image is then used in conjunction with a plurality of like images to determine the three-dimensional structure of the particle of interest. In preferred embodiments, this three-dimensional structure determination step includes an alignment step in which the orientation of the images employed in the structural determination is first determined based on the heavy atom cluster projections. The subject methods are particularly suited for use in determining the three-imensional structure of biological particles, e.g. proteins.